The black and white spotting pattern observed in piebald (s) mice results from abnormal neural crest development due to a mutation in endothelin receptor B (EDNRB). The severity and distribution of the pigment patterns are vastly different in two inbred strains carrying the s mutation (Mayers/s and C3H s/s). We hypothesized that additional genes may be responsible for coordinating the differences in patterning observed. Quantitative genetic analysis of backcross progeny from these two strains identified four genetic modifiers located on Chromosomes 2, 5, 8 and 10. The modifier on Chromosome 10 increases the dorsal spotting 2-fold more than ventral spotting (19.7% vs 9.1%, p < 0.0001), suggesting this modifier has spatial or temporal affects on pigment patterning. Analysis of mapping data implicates Steel (mast cell growth factor) as a candidate gene for this locus. Sequence comparison of cDNA isolates did not indicate any differences in the coding region, however differences in the level of steady state mRNA in adult tissues was observed by Northern blot analyses. Comparison of the genomic structure of the Steel gene demonstrated 12/12 restriction enzymes showing differences in the size of DNA fragments, however no differences were observed in two un-linked genes, EDNRB and endothelin 3. These results suggest the increased dorsal spotting observed in the Mayer strain of s mice is due to a mutation that alters the Steel expression pattern.We have tested this hypothesis by using crosses between the Mayer strain and Steel null mice. Consistent with our hypothesis, the Mayer allele at Steel cannot complement a Steel null mutation and results in increased dorsal spotting. Future studies using embryonic reconstitution experiments and expression pattern studies will also be explored to determine the molecular alterations and interactions at each modifier locus.